Analogue pigment studies of chromophore-protein interactions in metarhodopsins

Several analogue pigments have been prepared containing retinals altered at the cyclohexyl ring or proximal to the aldehyde group in order to examine the role of the chromophore in the formation of the metarhodopsin I and II states of visual pigments. Deletion of the 13-methyl group on the isoprenoid chain did not affect metarhodopsin formation. However, analogue pigments containing chromophores with modified rings did not show the typical absorption changes associated with the metarhodopsin transitions of native or regenerated rhodopsins. In particular, 4-hydroxyretinal pigments did not show clear transitions between the metarhodopsin I and metarhodopsin II states. Pigment formed with an acyclic retinal showed no evidence by absorption spectroscopy of metarhodopsin formation. A retinal altered by substitution of a five-membered ring containing a nitroxide required a more acidic pH than the native pigment for formation of the metarhodopsin II state. ESR data suggest that the ring remains buried within the protein through the metarhodopsin II state. However, the Schiff base linkage is susceptible to hydrolysis of hydroxylamine in the metarhodopsin II state. These data indicate that (1), in the transition from rhodopsin to metarhodopsin II, major protein conformational changes are occurring near the lysine-retinal linkage whereas the ring portion of the chromophore remains deeply buried within the protein and (2) pigment absorptions characteristic of the metarhodopsin I and II states may be due to specific protein-chromophore interactions near the region of the chromophore ring.     

Structural studies of metarhodopsin II,the activated form of the G-protein coupled receptor,rhodopsin

The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin. In particular, activation opens a groove on the surface of the receptor that could bind the N-terminal helix of the G protein, transducin alpha.     

Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.     

Light-induced interaction between rhodopsin and the GTP-binding protein. Metarhodopsin II is the major photoproduct involved

We have previously described [H, Kühn et al. (1981) Proc. Natl Acad. Sci. USA, 78, 6873-6877] a light-induced scattering change ('binding signal') associated with a stoichiometric binding between photoexcited rhodopsin and a peripheral membrane protein, the GTP-binding protein, in bovine rod outer segment suspensions. We have attempted here to identify the rhodopsin intermediate R* which is responsible for this interaction, by studying its dependence on pH, temperature and ionic strength. The results strongly suggest that the active state is metarhodopsin II (M II). 1. The initial phase of the binding signal is slightly slower than the formation of metarhodopsin II (2-37 degrees C, pH 5.5-9). 2. The kinetics of the decay of the active rhodopsin state are similar to those of the metarhodopsin II leads to metarhodopsin III transition (37 degrees C, pH 7.3). 3. All conditions which lead to light-induced binding of the GTP-binding protein to R* also lead to the formation of M II. At 2 degrees C, pH 8.3, in particular where no M II is formed in the absence of GTP-binding protein, binding signals and light-induced attachment of the GTP-binding protein to the membrane are still observed. Consistently, addition of GTP-binding protein to a suspension of extracted membranes bleached at 2 degrees C (pH 8.3) shifts the metarhodopsin I in equilibrium metarhodopsin II equilibrium towards metarhodopsin II. The shift is reversed by GTP, which dissociates the rhodopsin--GTP-binding protein complex. 4. At low ionic strength, where the GTP-binding protein is soluble in the dark (instead of being associated to the membrane as in the above experiments) M II still induces the binding whereas M I does not, indicating a much lower affinity of the GTP-binding protein for MI.     

Structural changes in lumirhodopsin and metarhodopsin I studied by their photoreactions at 77 K

The functional process of rhodopsin is initiated by cis-trans photoisomerization of the retinal chromophore. One of the primary intermediates, bathorhodopsin (Batho), is stable at 77 K, and structural changes in Batho are limited around the chromophore. Then, relaxation of Batho leads to helix opening at the cytoplasmic surface in metarhodopsin II (Meta II), which allows activation of a G protein transducin. Two intermediates, lumirhodopsin (Lumi) and metarhodopsin I (Meta I), appear between Batho and Meta II, and can be stabilized at 200 and 240 K, respectively. A photoaffinity labeling experiment reported that formation of Lumi accompanied flip-over of the beta-ionone ring of the retinal chromophore so that the ring portion was attached to Ala169 of helix IV [Borhan, B., Souto, M. L., Imai, H., Shichida, Y., and Nakanishi, K. (2000) Science 288, 2209-2212]. According to the crystal structure of bovine rhodopsin, the distance between the labeled C3 atom of the chromophore and Ala169 was >15 A [Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739-745]. These facts suggest that global protein structural changes such as helix motions take place in Lumi. In the study presented here, Lumi and Meta I are illuminated at 77 K, and protein structural changes are probed by Fourier transform infrared (FTIR) spectroscopy. We found that Lumi can be photoconverted to rhodopsin at 77 K from the IR spectral analysis of the photoproducts of Lumi. In contrast, more complex spectra were obtained for the photoproducts of Meta I at 77 K, implying that the protein structure of Meta I is considerably altered so as not to be reverted to the original state at 77 K. Thus, these photoreaction experiments with Lumi and Meta I at 77 K suggested the presence of global protein structural changes in the process between them. We concluded that the helix motions do not occur at Lumi, but at Meta I, and the flip-over of the beta-ionone ring reported by the photoaffinity labeling takes place through the specific reaction channel without a change in the global structure.     

Structural and functional role of helices I and II in rhodopsin. A novel interplay evidenced by mutations at Gly-51 and Gly-89 in the transmembrane domain

The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced lysine. Increased formation of non-active forms of rhodopsin, like metarhodopsin III, may have some influence in the molecular defect underlying retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of rhodopsin.     

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.     

Metarhodopsin control by arrestin,light-filtering screening pigments,and visual pigment turnover in invertebrate microvillar photoreceptors

The visual pigments of most invertebrate photoreceptors have two thermostable photo-interconvertible states, the ground state rhodopsin and photo-activated metarhodopsin, which triggers the phototransduction cascade until it binds arrestin. The ratio of the two states in photoequilibrium is determined by their absorbance spectra and the effective spectral distribution of illumination. Calculations indicate that metarhodopsin levels in fly photoreceptors are maintained below ~35% in normal diurnal environments, due to the combination of a blue-green rhodopsin, an orange-absorbing metarhodopsin and red transparent screening pigments. Slow metarhodopsin degradation and rhodopsin regeneration processes further subserve visual pigment maintenance. In most insect eyes, where the majority of photoreceptors have green-absorbing rhodopsins and blue-absorbing metarhodopsins, natural illuminants are predicted to create metarhodopsin levels greater than 60% at high intensities. However, fast metarhodopsin decay and rhodopsin regeneration also play an important role in controlling metarhodopsin in green receptors, resulting in a high rhodopsin content at low light intensities and a reduced overall visual pigment content in bright light. A simple model for the visual pigment–arrestin cycle is used to illustrate the dependence of the visual pigment population states on light intensity, arrestin levels and pigment turnover.     

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state ²H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific ²H labels have been introduced into the methyl groups of retinal and solid-state ²H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent ²H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu¹²² and Trp²⁶⁵ within the binding pocket. The solid-state ²H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.     

Transition of rhodopsin into the active metarhodopsin II state opens a new light-induced pathway linked to Schiff base isomerization

Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced by absorption of green light, leads to active metarhodopsin II, in which the Schiff base is intact but deprotonated. The subsequent metabolic retinoid cycle starts with Schiff base hydrolysis and release of photolyzed all-trans-retinal from the active site and ends with the uptake of fresh 11-cis-retinal. To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UV-visible spectroscopy. A light-induced shortcut of the retinoid cycle, as it occurs in other retinal proteins, is not observed. The predominantly formed illumination product contains all-trans-retinal, although the spectra reflect Schiff base reprotonation and protein deactivation. By its kinetics of formation and decay, its low temperature photointermediates, and its interaction with transducin, this illumination product is identified as metarhodopsin III. This species is known to bind all-trans-retinal via a reprotonated Schiff base and forms normally in parallel to retinal release. We find that its generation by light absorption is only achieved when starting from active metarhodopsin II and is not found with any of its precursors, including metarhodopsin I. Based on the finding of others that metarhodopsin III binds retinal in all-trans-C(15)-syn configuration, we can now conclude that light-induced formation of metarhodopsin III operates by Schiff base isomerization ("second switch"). Our reaction model assumes steric hindrance of the retinal polyene chain in the active conformation, thus preventing central double bond isomerization.     

Diffusible ligand all-trans-retinal activates opsin via a palmitoylation-dependent mechanism

In rhodopsin's function as a photoreceptor, 11-cis-retinal is covalently bound to Lys(296) via a protonated Schiff base. 11-cis/all-trans photoisomerization and relaxation through intermediates lead to the metarhodopsin II photoproduct, which couples to transducin (G(t)). Here we have analyzed a different signaling state that arises from noncovalent binding of all-trans-retinal (atr) to the aporeceptor opsin and enhances the very low opsin activity by several orders of magnitude. Like with metarhodopsin II, coupling of G(t) to opsin-atr is sensitive to competition by synthetic peptides from the COOH termini of both G(t)alpha and G(t)gamma. However, atr does not compete with 11-cis-retinal incorporation into the Lys(296) binding site and formation of the light-sensitive pigment. Blue light illumination fails to photorevert opsin-atr to the ground state. Thus noncovalently bound atr has no access to the light-dependent binding site and reaction pathway. Moreover, in contrast to light-dependent signaling, removal of the palmitoyl anchors at Cys(322) and Cys(323) in the rhodopsin COOH terminus impairs the atr-stimulated activity. Repalmitoylation by autoacylation with palmitoyl-coenzyme A restores most of the original activity. We hypothesize that the palmitoyl moieties are part of a second binding pocket for the chromophore, mediating hydrophobic interactions that can activate a large part of the catalytic receptor/G-protein interface.     

Structure of rhodopsin and the metarhodopsin I photointermediate

The structure of the visual pigment rhodopsin in the dark state was first investigated by electron microscopy (EM). More recently, rhodopsin has been crystallised in two different space groups--a tetragonal P4(1) crystal form and a trigonal P3(1) packing arrangement. The structures of the pigment, determined by X-ray crystallography from these two crystal forms, show many similarities, but also significant differences. These differences are most extensive in the G-protein-binding region of the cytoplasmic surface, where the location of the loop between helices 5 and 6 is highly variable. A combination of EM and spin labelling suggests that this loop adopts the native conformation in the P3(1) crystal form. The X-ray structures also show the location of structural water molecules that are important for colour tuning, stabilisation of the ground state and receptor activation, and act as a template for modelling other G-protein-coupled receptors. A major current focus of structural work on rhodopsin is investigation of the activated state of the receptor. After careful spectroscopic characterisation of light activation in two-dimensional crystals, a map of the metarhodopsin I intermediate was obtained by EM from two-dimensional crystals. In addition, NMR studies are providing information about the structure of activated states of rhodopsin. In the future, structural information will show how rhodopsin becomes activated and how it couples to downstream signalling pathways.     

Photochemical functionality of rhodopsin-phospholipid recombinant membranes

Purified rhodopsin was incorporated into phospholipid bilayers to give recombinant membranes. The photochemical functionality in these systems was examined by low-temperature spectroscopy and by kinetic spectrophotometry. Changes in the absorption spectra of glycerol-water mixtures of rhodopsin-egg phosphatidylcholine and rhodopsin-asolectin recombinants were monitored after the sample was cooled to -196 degrees C, presented with light of wavelength greater than 440 nm, and then warmed gradually to room temperature. Absorption characteristics indicative of the spectral intermediates prelumirhodopsin, lumirhodopsin, metarhodopsin I, and metarhodopsin II were observed. The kinetics of the metarhodopsin I -o metarhodopsin II transition in these recombinants was studied by flash photolytic observation of the decay of meta I and the formation of meta II. Recombinants prepared from unsaturated phospholipids, e.g., asolectin, egg phosphatidylcholine, egg phosphatidylethanolamine, and dioleoylphosphatidylcholine, showed first-order kinetics for the transition with rates comparable to that of rod outer segment membranes. Recombinants prepared from saturated phosphatidylcholines have a retarded rate of conversion from meta I to meta II and are considered to be nonfunctional. The photochemical functionality of rhodopsin-phospholipid recombinants is dependent upon the presence of phospholipid unsaturation and the fluidity of the phospholipid hydrocarbon chains, and is independent of the polar head group of the phospholipid.     

Light Activation of Rhodopsin: Insights from Molecular Dynamics Simulations Guided by Solid-State NMR Distance Restraints

Structural restraints provided by solid-state NMR measurements of the metarhodopsin II intermediate are combined with molecular dynamics simulations to help visualize structural changes in the light activation of rhodopsin. Since the timescale for the formation of the metarhodopsin II intermediate (> 1 ms) is beyond that readily accessible by molecular dynamics, we use NMR distance restraints derived from ¹³C dipolar recoupling measurements to guide the simulations. The simulations yield a working model for how photoisomerization of the 11-cis retinylidene chromophore bound within the interior of rhodopsin is coupled to transmembrane helix motion and receptor activation. The mechanism of activation that emerges is that multiple switches on the extracellular (or intradiscal) side of rhodopsin trigger structural changes that converge to disrupt the ionic lock between helices H3 and H6 on the intracellular side of the receptor.     

Conformational change of helix G in the bacteriorhodopsin photocycle: investigation with heavy atom labeling and x-ray diffraction.

According to the current structural model of bacteriorhodopsin, Ile222 is located at the cytoplasmic end of helix G. We labeled the single cysteine of the site-directed mutant Ile222 --> Cys with p-chloromercuribenzoic acid and determined the position of the labeled mercury by x-ray diffraction in the unphotolyzed state, and in the MN photointermediate accumulated in the presence of guanidine hydrochloride at pH 9.5. According to the difference Fourier maps between the MN intermediate and the unphotolyzed state, the structural change in the MN intermediate was not affected by mercury labeling. The difference Fourier map between the labeled and the unlabeled I222C gave the position of the mercury label. This information was obtained for both the unphotolyzed state and the MN intermediate. We found that the position of the mercury at residue 222 is shifted by 2.1 +/- 0.8 A in the MN intermediate. This agrees with earlier results that suggested a structural change in the G helix. The movement of the mercury label is so large that it must originate from a cooperative conformational change in the helix G at its cytoplasmic end, rather than from displacement of residue 222. Because Ile222 is located at the same level on the z coordinate as Asp96, the structural change in the G helix could have the functional role of perturbing the environment and therefore the pKa of this functionally important aspartate.     

Resonance Raman studies of bovine metarhodopsin I and metarhodopsin II

The resonance Raman spectra of bovine metarhodopsin I and metarhodopsin II have been measured. The spectra are compared with model chromophore resonance Raman data. It was found that metarhodopsin I is linked to opsin via a protonated Schiff base linkage, whereas metarhodopsin II is linked by an unprotonated Schiff base. A recent suggestion that the chromophore of metarhodopsin II is retinal is explicitly disproved. The chromophores of both metarhodopsins are found to have an essentially all-trans conformation. The basic mechanism for color regulation in both forms appears to be electron delocalization. The data tend to support the model of cis-trans isomerization as the primary mechanism for vision. Also, the conclusions and inferences of this work on energy uses and storage by rhodopsin in neural generation are discussed.     

Linkage between the intramembrane H-bond network around aspartic acid 83 and the cytosolic environment of helix 8 in photoactivated rhodopsin

Understanding the coupling between conformational changes in the intramembrane domain and at the membrane-exposed surface of the bovine photoreceptor rhodopsin, a prototypical G protein-coupled receptor (GPCR), is crucial for the elucidation of molecular mechanisms in GPCR activation. Here, we have combined Fourier transform infrared (FTIR) and fluorescence spectroscopy to address the coupling between conformational changes in the intramembrane region around the retinal and the environment of helix 8, a putative cytosolic surface switch region in class-I GPCRs. Using FTIR/fluorescence cross-correlation we show specifically that surface alterations monitored by emission changes of fluorescein bound to Cys316 in helix 8 of rhodopsin are highly correlated with (i) H-bonding to Asp83 proximal of the retinal Schiff base but not to Glu122 close to the beta-ionone and (ii) with a metarhodopsin II (MII)-specific 1643 cm(-1) IR absorption change, indicative of a partial loss of secondary structure in helix 8 upon MII formation. These correlations are disrupted by limited C-terminal proteolysis but are maintained upon binding of a transducin alpha-subunit (G(talpha))-derived peptide, which stabilizes the MII state. Our results suggest that additional C-terminal cytosolic loop contacts monitored by an amide II absorption at 1557 cm(-1) play a functionally crucial role in keeping helix 8 in the position in which its environment is strongly coupled to the retinal-binding site near the Schiff base. In the intramembrane region, this coupling is mediated by the H-bonding network that connects Asp83 to the NPxxY(x)F motif preceding helix 8.     

Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin

Isomerization of the 11-cis retinal chromophore in the visual pigment rhodopsin is coupled to motion of transmembrane helix H6 and receptor activation. We present solid-state magic angle spinning NMR measurements of rhodopsin and the metarhodopsin II intermediate that support the proposal that interaction of Trp265(6.48) with the retinal chromophore is responsible for stabilizing an inactive conformation in the dark, and that motion of the beta-ionone ring allows Trp265(6.48) and transmembrane helix H6 to adopt active conformations in the light. Two-dimensional dipolar-assisted rotational resonance NMR measurements are made between the C19 and C20-methyl groups of the retinal and uniformly 13C-labeled Trp265(6.48). The retinal C20-Trp265(6.48) contact present in the dark-state of rhodopsin is lost in metarhodopsin II, and a new contact is formed with the C19 methyl group. We have previously shown that the retinal translates 4-5 A toward H5 in metarhodopsin II. This motion, in conjunction with the Trp-C19 contact, implies that the Trp265(6.48) side-chain moves significantly upon rhodopsin activation. NMR measurements also show that a packing interaction in rhodopsin between Trp265(6.48) and Gly121(3.36) is lost in metarhodopsin II, consistent with H6 motion away from H3. However, a close contact between Gly120(3.35) on H3 and Met86(2.53) on H2 is observed in both rhodopsin and metarhodopsin II, suggesting that H3 does not change orientation significantly upon receptor activation.     

Bleaching kinetics of artificial visual pigments with modifications near the ring-polyene chain connection

Absorbance difference spectra were recorded at 20 degrees C from 30 ns to milliseconds after photolysis of lauryl maltoside suspensions of artificial visual pigments derived from 9-cis isomers of 5-ethylretinal, 8,16-methanoretinal (a 6-s-trans-bicyclic analogue), or 5-demethyl-8-methylretinal. In all three pigments, the earliest intermediate that was detected had the characteristics of a mixture of bathorhodopsin and a blue-shifted intermediate, BSI, which is the first decay product of bathorhodopsin in bovine rhodopsin. The first decays resolved on the nanosecond time scale were the formation of the lumirhodopsin analogues. Subsequent decays were able to be fit with a mechanistic scheme which has been shown to apply to both membrane and detergent suspensions of rhodopsin. Large increases were seen in the amount of metarhodopsin I which appeared after photolysis of 5-ethylisorhodopsin and the bicyclic isorhodopsin analogue, while 5-demethyl-8-methylisorhodopsin more closely followed native rhodopsin in decaying through meta I380, a 380 nm absorbing precursor to metarhodopsin II. In addition to forming more metarhodopsin I, the bicyclic analogue stabilized the metarhodopsin I-metarhodopsin II equilibrium similarly to what has been previously reported for 9-demethylrhodopsin in detergent, introducing the possibility that the bicyclic analogue could similarly be defective in transducin activation. These observations support the idea that long after initial photolysis, structural details of the retinylidene chromophore continue to play a decisive role in processes leading to the activated form, metarhodopsin II.     

Tautomeric Forms of Metarhodopsin

Light isomerizes the chromophore of rhodopsin, 11-cis retinal (formerly retinene), to the all-trans configuration. This introduces a succession of unstable intermediates—pre-lumirhodopsin, lumirhodopsin, metarhodopsin —in which all-trans retinal is still attached to the chromophoric site on opsin. Finally, retinal is hydrolyzed from opsin. The present experiments show that metarhodopsin exists in two tautomeric forms, metarhodopsins I and II, with λ_max 478 and 380 mµ. Metarhodopsin I appears first, then enters into equilibrium with metarhodopsin II. In this equilibrium, the proportion of metarhodopsin II is favored by higher temperature or pH, neutral salts, and glycerol. The change from metarhodopsin I to II involves the binding of a proton by a group with pK 6.4 (imidazole?), and a large increase of entropy. Metarhodopsin II has been confused earlier with the final mixture of all-trans retinal and opsin (λ_max 387 mµ), which it resembles in spectrum. These two products are, however, readily distinguished experimentally.